Palovarotene Inhibits Osteochondroma Formation in a Mouse Model of Multiple Hereditary Exostoses

Abstract

Multiple hereditary exostoses (MHE), also known as multiple osteochondromas (MO), is an autosomal dominant disorder characterized by the development of multiple cartilage-capped bone tumors (osteochondromas). The large majority of patients with MHE carry loss-of-function mutations in the EXT1 or EXT2 gene, which encodes a glycosyltransferase essential for heparan sulfate (HS) biosynthesis. Increasing evidence suggests that enhanced bone morphogenetic protein (BMP) signaling resulting from loss of HS expression plays a role in osteochondroma formation in MHE. Palovarotene (PVO) is a retinoic acid receptor γ selective agonist, which is being investigated as a potential drug for fibrodysplasia ossificans progressiva (FOP), another genetic bone disorder with features that overlap with those of MHE. Here we show that PVO inhibits osteochondroma formation in the Fsp1^Cre ;Ext1^flox/flox model of MHE. Four-week daily treatment with PVO starting at postnatal day (P) 14 reduced the number of osteochondromas that develop in these mice by up to 91% in a dose-dependent manner. An inhibition of long bone growth observed in animals treated from P14 was almost entirely abrogated by delaying the initiation of treatment to P21. We also found that PVO attenuates BMP signaling in Fsp1^Cre ;Ext1^flox/flox mice and that aberrant chondrogenic fate determination of Ext1-deficient perichondrial progenitor cells in these mice is restored by PVO. Together, the present data support further preclinical and clinical investigations of PVO as a potential therapeutic agent for MHE. © 2017 American Society for Bone and Mineral Research.

Keywords: EXT1; HEPARAN SULFATE; MULTIPLE HEREDITARY EXOSTOSIS (MHE); MULTIPLE OSTEOCHONDROMAS (MO); PALOVAROTENE.

Figure 1. PVO suppresses osteochondroma formation in Fsp1^Cre;Ext1^flox/flox mice

Fsp1^Cre;Ext1^flox/flox mice were treated by daily oral gavage with 0.27, 0.88 or 1.76 mg/kg of PVO or vehicle (Vehicle) and the effects on osteochondroma formation and bone growth were analyzed at the end of the treatments. (A–D) Fsp1^Cre;Ext1^flox/flox mice were treated from P14 to P42 (Experiment 1). (E–H) Fsp1^Cre;Ext1^flox/flox mice were treated from P21 to P42 (Experiment 2). (A and E) The total number of osteochondromas in five major limb bones (ulna, radius, humerus, tibia, femur) of the right hemi-skeleton per animal. (B and F) The total number of osteochondromas in 12 ribs of the right hemi-skeleton per animal. (C and G) The longitudinal length of the tibia. (D and H) The longitudinal length of the femur. In Experiment 1, the mean number of osteochondromas in the 0.27, 0.88 or 1.76 mg/kg/day groups in comparison with the vehicle group was reduced by 56.8%, 84.6%, and 91.1%, respectively, in long bones (A) and by 57.5%, 81.8%, and 90.8%, respectively, in ribs (B). With this treatment protocol (i.e., P14–42), the longitudinal length of long bones was reduced by up to 21.8% (C and D). In Experiment 2, the mean number of osteochondromas in the 0.27, 0.88 or 1.76 mg/kg/day groups in comparison with the vehicle group was reduced by 32.0%, 67.8%, and 88.4%, respectively, in long bones (E) and by 34.1%, 54.6%, and 77.6%, respectively, in ribs (F). With this treatment protocol (i.e., P21–42), PVO showed no inhibitory effect on the longitudinal growth of long bones (G and H). See also the Supplementary Table 1 for the summary of all data from these drug treatment experiments, including the data on the ulna, radius, and humerus, which are not shown in this figure. * p<0.05, *** p<0.001 by one-way ANOVA followed by Bonferroni’s post hoc test. n.s., not significant. (I) Representative images of hindlimbs and ribs in the indicated treatment groups. Scale bars, 0.1 mm.

Figure 2. Histological assessment of the effect of PVO

Fsp1^Cre;Ext1^flox/flox (CKO) and wild-type (WT) mice, treated with 1.76 mg/kg/day PVO or vehicle from P21 to P42 (Experiment 2), were histologically examined at P42. (A and B) Safranin O-stained sections of the femur (A–D) and the 10th rib (E–H). Right images in each panel show high-magnification images of the perichondrium (PC) and periosteum (PO). Note that there are numerous abnormal chondrocyte clusters and overgrowth of cartilage in vehicle-treated Fsp1^Cre;Ext1^flox/flox mice (C and G). In PVO-treated Fsp1^Cre;Ext1^flox/flox mice, such lesions mostly disappear (D and H), although there are occasional ectopic clusters of chondrocytes (arrowheads). (I) Effects of PVO on growth plate organization. Safranin O-stained sections of the distal femur growth plate reveal an irregular morphology of the growth plate and a mild disorganization of chondrocytic columnization in vehicle-treated Fsp1^Cre;Ext1^flox/flox mice (CKO/Vehicle; see also Fig. 2C). PVO treatment partially restores the normal organization of the growth plate (CKO/PVO). Data shown are representative images. Each analysis was performed on at least 5 animals per genotype. (J) Effects of PVO on chondrocyte proliferation. After 3-week treatment with PVO or vehicle, mice at P42 were analyzed by BrdU labeling. The ratio of proliferating cells was determined by dividing the number of BrdU-positive cells by total number of DAPI-stained nuclei in the proliferating zone of the femoral growth plate (Growth plate; n = 5 animals/genotype/treatment) and in the cartilage cap of osteochondromas (Osteochondroma; n = 27 discrete osteochondromas/treatment). Means are shown as horizontal bars. Two-way ANOVA followed by Bonferroni’s post hoc test was used for the growth plate data and student’s t test for the osteochondroma data; n.s., not significant. Scale bars, 0.1 mm (A–D and I); 0.2 mm (E–H).

Figure 3. PVO restores normal fate determination of Ext1-deficient progenitor cells in the perichondrium

Fsp1^Cre;R26^tdTomato mice (WT) and Fsp1^Cre;R26^tdTomato;Ext1^flox/flox (CKO) mice were treated by daily oral gavage of 1.76 mg/kg PVO or vehicle starting at P21. At P31, mice were sacrificed and frozen sections of the 10th rib were examined for the localization of tdTomato⁺ cells, which are cells that have undergone Fsp1^Cre-mediated recombination and their progeny. Sections were also stained with anti-CD44 (CD44) and DAPI or anti-type II collagen antibody (Col2) and DAPI to determine the differentiation state of tdTomato⁺ cells. Smaller images on the lower right of each panel represent high-magnification views of the perichondrium/periosteum. (A) Fsp1^Cre;R26^tdTomato (WT) mice treated with vehicle. (B) Fsp1^Cre;R26^tdTomato;Ext1^flox/flox (CKO) mice treated with vehicle. (C) Fsp1^Cre;R26^Tomato;Ext1^flox/flox (CKO) mice treated with PVO. Note that tdTomato⁺ progenitor cells in WT mice (A) also express CD44 (thus yellow signals in the upper left image) but not Col2 (no yellow signals in the lower left image), which indicates that Fsp1-expressing progenitor cells maintain the property of progenitor cells and that their progeny contribute primarily to osteoblastic cells. In contrast, in CKO mice (B), Ext1-deficient progenitor cells contribute to Col2⁺ chondrocytes forming ectopic clusters of chondrocytes (white arrowheads). CKO mice treated with PVO (C) show reversion of tdTomato⁺ progenitor cells to the normal pattern of fate determination. Scale bars, 0.1 mm.

Figure 4. PVO attenuates enhanced BMP signaling in Ext1-deficient perichondrium

Fsp1^Cre;R26^tdTomato;Ext1^flox/flox (CKO) mice were treated by daily oral gavage of 1.76 mg/kg PVO or vehicle for 5 days starting at P21. At P26, these mice as well as vehicle-treated Fsp1^Cre;R26^tdTomato (WT) mice were sacrificed, and frozen sections of the 10th ribs were stained with anti-pSmad1/5/8 antibody (green). Fsp1^Cre-mediated recombination was monitored by tdTomato (red). (A) pSmad1/5/8 expression in the perichondrium. (B) pSmad1/5/8 expression in the periosteum. Note that only low levels of pSmad1/5/8 are seen in the perichondrium and periosteum of vehicle-treated WT mice (WT/Vehicle), whereas Smad1/5/8 is strongly phosphorylated in vehicle-treated CKO mice (CKO/Vehicle). In PVO-treated CKO mice, Smad1/5/8 phosphorylation is attenuated to the WT level (CKO/PVO). Data shown are representative images. Each analysis was performed on at least 3 animals per genotype. Scale bars, 50 μm (A and B). (C) Ext1-deficient (Ext1^−/−) and wild-type (WT) PDPCs in monolayer cultures were pretreated with 1 μM PVO in DMSO or DMSO only for 48 h. Then, cells were incubated with 10 ng/ml BMP2 for 30 min and lysed. Cell lysates were immunoblotted with antibodies to pSmad1/5/8, total Smad1/5/8, and α-tubulin. This experiment was performed three times with similar results.